ADCs have emerged as a promising class of targeted therapeutics with significant potential to improve clinical efficacy and tolerability over antibody therapy or traditional chemotherapy. Clinically useful ADCs are capable of antigen-specific delivery of highly potent cytotoxic drugs to tumor cells. Monoclonal antibody moiety of ADCs can specifically recognize cell surface antigens which are substantially more elevated in tumor cells than healthy cells, thus decreasing non-specific uptake and increasing specific update of conjugated drugs by tumor cells. Recent clinical data have led to the commercialization of two FDA-approved ADCs products, including brentuximab vedotin: an anti-CD30 monoclonal antibody conjugate, and Ado-trastuzumab emtansine: an anti-HER2 monoclonal antibody conjugate. A third marketed ADC is gemtuzumab ozogamicin, an anti-CD33 monoclonal antibody conjugate, is commercially available in Japan.
The approach by which drugs attach to an antibody (i.e., conjugation) is an important aspect of ADC development. All three referenced commercial ADC products utilize conventional non-specific conjugation method. Brentuximab vedotin is produced by the modification of native cysteine side chain thiols in solvent-exposed disulfides, whereas ado-trastuzumab emtansine and gemtuzumab ozogamicin are made via modification of surface lysine side chain amines. These non-specific conjugation methods have resulted in heterogeneous ADC mixtures.
In order to improve therapeutic index and pharmacokinetics of ADCs, cysteine-based site-specific ADCs have recently been developed to generate more homogeneous drug products with greater control over drug attachment sites. Unpaired cysteine residues have long been introduced into proteins for site-specific labeling and drug conjugation. See: Lyons et al., Protein Eng. 3, 703-708 (1990); Zheng et al., Biochemistry, 30, 9125-9132 (1991); Stimmel, et al., J. Biol. Chem. 275, 30445-30450 (2000); Junutula et al., Nat. Biotechnol., 26, 925-932 (2008); Voynov et al., Bioconjug. Chem. 21, 385-392 (2010); and Shen et al., Nat. Biotechnol., 30, 184-189 (2012). These engineered cysteine residues are typically located on the surface of a protein, and do not alter protein structure and function. It has been recently shown that cysteine-based site-specific ADCs possess improved therapeutic index and reduced toxicity over conventional Cys conjugates and Lys conjugates. See: Junutula et al., Nat. Biotechnol., 26, 925-932 (2008); Junutula et al., Clin. Cancer Res. 16, 4769-47788 (2010); Shen et al., Nat. Biotechnol., 30, 184-189 (2012); and Kung et al., Blood 122, 1455-1463 (2013).
Cysteine-based site-specific ADCs, however, introduce complexity into the drug conjugation process. When produced in mammalian cells, the thiol group(s) of unpaired cysteine residues of cysteine mutant antibody has been found to form disulfides with other cysteines (cysteinylation) or glutathione (glutathionylation) (Junutula, Raab et al. 2008, Chen, Nguyen et al. 2009). These post-translational modifications are called cysteine-capping or Cys-capping. This cysteine-capping creates thiol linked blocking groups which prevent or inhibit conjugation, and thus prior to drug conjugation the thiol group needs to be regenerated through a partial reduction step with reducing agents. Since this treatment also reduces the antibody inter-chain disulfides (also known as “paired” cycteines) those reduced antibody inter-chain disulfides must then be reformed. This is accomplished in a re-oxidation process including dialyzing out reducing agents, cysteine or glutathione, and treating with oxidation reagents. This reduction and reoxidation potentially introduces disulfide shuffling (also called disulfide scrambling, see dashed oval below for illustration) and twisting on the antibody. A twisted antibody can adversely affect protein folding and protein quality, and also cause issues such as poorer PK for resulting ADCs. This phenomenon is shown in FIG. 17.
The underlining mechanism for these “natural” cysteinylation and glutathionylation cappings are unclear. Since both modifications involve forming disulfide bond, it has long been speculated that these modifications may take place in the lumen of endoplasmic reticulum (ER) where disulfide bond formation occurs. It is well known that ER lumen is more oxidized than cytosol (Hwang, Sinskey et al. 1992), due to a highly-conserved oxidation molecular pathway (Frand, Cuozzo et al. 2000, Sevier and Kaiser 2006). Flavin-containing membrane-protein Ero1 (Frand and Kaiser 1998, Pollard, Travers et al. 1998) exploits oxidation power of oxygen to introduce disulfide bonds within itself, then transfers disulfide bond to protein disulfide isomerase (PDI) which can pass it onto extracellular proteins (Tu, Ho-Schleyer et al. 2000). Alternative Ero1-independent oxidation pathways, such as quiescin sulphydryl oxidase/Erv superfamily and vitamin K epoxide reductase, also contribute to disulfide bond formation in mammalian cells (Margittai and Banhegyi 2010, Sevier 2010). GSH is present in ER lumen due to either a transporter (Hwang, Sinskey et al. 1992, Banhegyi, Lusini et al. 1999) or pores (Le Gall, Neuhof et al. 2004) in the membrane. Cys is also presumably present due to a transport activity (Hwang, Sinskey et al. 1992). Therefore an oxidative ER lumen plus the presence of GSH and Cys has made ER lumen a reasonable place for glutathionylation or cysteinylation. However, no conclusive evidence exists in supporting this hypothesis.